A. Congestive Heart Failure
Congestive heart failure (CHF) affects nearly 5 million Americans with over 500,000 new cases diagnosed annually. By definition, CHF is a clinical syndrome in which heart disease reduces cardiaC output, increases venous pressures, and is accompanied by molecular abnormalities that cause progressive deterioration of the failing heart (From; Heart Failure: Pathophysiology, Molecular Biology, and Clinical Management, Katz, AM, Lippincott Williams and Wilkins, 2000). Despite decades of research a detailed understanding of the causes of CHF are still unclear. However, scientific and clinical findings clearly demonstrate that an early phase of the disease process consists of a maladaptive response of the myocardium to stress known as ‘cardiac hypertrophy’ (also, ‘hypertrophic cardiomyopathy’). Chronic overload on the heart in the setting of unremitting hypertension, valve disease, or tissue damage (myocardial infarction) results in a hypertrophic growth response which is initially adaptive in so far as cardiac output is temporarily restored but gradually becomes maladaptive over time resulting in decreased contractile function, cardiac dilatation and failure. Because the 5-year survival rate, once heart failure becomes symptomatic, is less that 50%, any definition of heart failure that does not consider the molecular processes that accelerate myocardial hypertrophy overlooks a major clinical feature of this syndrome.
Cell culture and small animal studies have clearly demonstrated that G-protein coupled receptors on cardiac myocytes are highly important regulators of cardiac contractile function and are also involved in the regulation of myocyte hypertrophy (for review see; Adams and Brown, Oncogene, 20, 1626-1634, 2001). In fact, the positive effects of ACE inhibitors for treatment of CHF in humans is thought to at least partially involve the reduction of maladaptive hypertrophy via indirect inhibition of angiotensin II receptor activation in the myocardium.
However, despite improvements in pharmacological therapies for CHF over the past ten years (ACE inhibitors, beta-blockers) only a 20-30% reduction in mortality has been demonstrated with current optimal therapies. In the future, development of better drugs and identification of new therapeutic targets will likely improve the clinical outcome of patients with CHF.
B. G Protein-Coupled Receptors
Although a number of receptor classes exist in humans, by far the most abundant and therapeutically relevant is represented by the G protein-coupled receptor (GPCR) class. It is estimated that there are some 30,000-40,000 genes within the human genome, and of these, approximately 2% are estimated to code for GPCRs.
GPCRs represent an important area for the development of pharmaceutical products: from approximately 20 of the 100 known GPCRs, approximately 60% of all prescription pharmaceuticals have been developed. For example, in 1999, of the top 100 brand name prescription drugs, the following drugs interact with GPCRs (the primary diseases and/or disorders treated related to the drug is indicated in parentheses):
Claritin ® (allergies)Prozac ® (depression)Vasotec ® (hypertension)Paxil ® (depression)Zoloft ® (depression)Zyprexa ® (psychotic disorder)Cozaar ® (hypertension)Imitrex ® (migraine)Zantac ® (reflux)Propulsid ® (reflux disease)Risperdal ® (schizophrenia)Serevent ® (asthma)Pepcid ® (reflux)Gaster ® (ulcers)Atrovent ® (bronchospasm)Effexor ® (depression)Depakote ® (epilepsy)Cardura ® (prostatic hypertrophy)Allegra ® (allergies)Lupron ® (prostate cancer) Zoladex ® (prostate cancer)Diprivan ® (anesthesia)BuSpar ® (anxiety)Ventolin ® (bronchospasm)Hytrin ® (hypertension)Wellbutrin ® (depression)Zyrtec ® (rhinitis)Plavix ® (MI/stroke)Toprol-XL ® (hypertension)Tenormin ® (angina)Xalatan ® (glaucoma)Singulair ® (asthma)Diovan ® (hypertension)Hamal ® (prostatic hyperplasia)(Med Ad News 1999 Data).
GPCRs share a common structural motif, having seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane [each span is identified by number, i.e., transmembrane-1 (TM-1), transmembrane-2 (TM-2), etc.]. The transmembrane helices are joined by strands of amino acids between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the exterior, or “extracellular” side, of the cell membrane [these are referred to as “extracellular” regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively]. The transmembrane helices are also joined by strands of amino acids between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the interior, or “intracellular” side, of the cell membrane [these are referred to as “intracellular” regions 1, 2 and 3 (IC-1, IC-2 and IC-3), respectively]. The “carboxy” (“C”) terminus of the receptor lies in the intracellular space within the cell, and the “amino” (“N”) terminus of the receptor lies in the extracellular space outside of the cell.
Generally, when a ligand binds with the receptor (often referred to as “activation” of the receptor), there is a Change in the conformation of the receptor that facilitates coupling between the intracellular region and an intracellular “G-protein.” It has been reported that GPCRs are “promiscuous” with respect to G proteins, i.e., that a GPCR can interact with more than one G protein. See, Kenakin, T., 43 Life Sciences 1095 (1988). Although other G proteins exist, currently, Gq, Gs, Gi, Gz and Go are G proteins that have been identified. Ligand-activated GPCR coupling with the G-protein initiates a signaling cascade process (referred to as “signal transduction”). Under normal conditions, signal transduction ultimately results in cellular activation or cellular inhibition. Although not wishing to be bound to theory, it is thought that the IC-3 loop as well as the carboxy terminus of the receptor interact with the G protein.
Gs-coupled GPCRs elevate intracellular cAMP levels. Gi-, Go-, or Gz-coupled GPCRs lower intracellular cAMP levels. Gq-coupled GPCRs elevate intracellular IP3 and Ca2+ levels.
There are also promiscuous G proteins, which appear to couple several classes of GPCRs to the phospholipase C pathway, such as G15 or G16 [Offermanns & Simon, J Biol Chem (1995) 270:15175-80], or chimeric G proteins designed to couple a large number of different GPCRs to the same pathway, e.g. phospholipase C [Milligan & Rees, Trends in Pharmaceutical Sciences (1999) 20:118-24].
The melanophore technology (see infra) is useful in interrogating the G-protein coupling of a GPCR and also for identifying whether a compound is a modulator of the GPCR.
Under physiological conditions, GPCRs exist in the cell membrane in equilibrium between two different conformations: an “inactive” state and an “active” state. A receptor in an inactive state is unable to link to the intracellular signaling transduction pathway to initiate signal transduction leading to a biological response. Changing the receptor conformation to the active state allows linkage to the transduction pathway (via the G-protein) and produces a biological response.
A receptor may be stabilized in an active state by a ligand or a compound such as a drug. Recent discoveries, including but not exclusively limited to modifications to the amino acid sequence of the receptor, provide means other than ligands or drugs to promote and stabilize the receptor in the active state conformation. These means effectively stabilize the receptor in an active state by simulating the effect of a ligand binding to the receptor. Stabilization by such ligand-independent means is termed “constitutive receptor activation” (see, e.g., PCT Application Number PCT/US98/07496 published as WO 98/46995 on 22 Oct. 1998; the disclosure of which is hereby incorporated by reference in its entirety).